The relationship between tumour dosimetry, response, and overall survival in patients with unresectable Neuroendocrine Neoplasms (NEN) treated with 177Lu DOTATATE (LuTate).

Peptide Receptor Radionuclide Therapy (PRRT) delivers targeted radiation to Somatostatin Receptor (SSR) expressing Neuroendocrine Neoplasms (NEN). We sought to assess the predictive and prognostic implications of tumour dosimetry with respect to response by 68 Ga DOTATATE (GaTate) PET/CT molecular imaging tumour volume of SSR (MITVSSR) change and RECIST 1.1, and overall survival (OS). METHODS: Patients with gastro-entero-pancreatic (GEP) NEN who received LuTate followed by quantitative SPECT/CT (Q-SPECT/CT) the next day (Jul 2010 to Jan 2019) were retrospectively reviewed. Single time-point (STP) lesional dosimetry was performed for each cycle using population-based pharmacokinetic modelling. MITVSSR and RECIST 1.1 were measured at 3-months post PRRT. RESULTS: Median of 4 PRRT cycles were administered to 90 patients (range 2-5 cycles; mean 27.4 GBq cumulative activity; mean 7.6 GBq per cycle). 68% received at least one cycle with radiosensitising chemotherapy (RSC). RECIST 1.1 partial response was 24%, with 70% stable and 7% progressive disease. Cycle 1 radiation dose in measurable lesions was associated with local response (odds ratio 1.5 per 50 Gy [95% CI: 1.1-2.0], p = 0.002) when adjusted by tumour grade and RSC. Median change in MITVSSR was -63% (interquartile range -84 to -29), with no correlation with radiation dose to the most avid lesion on univariable or multivariant analyses (5.6 per 10 Gy [95% CI: -1.6, 12.8], p = 0.133). OS at 5-years was 68% (95% CI: 56-78%). Neither baseline MITVSSR (hazard ratio 1.1 [95% CI: 1.0, 1.2], p = 0.128) nor change in baseline MITVSSR (hazard ratio 1.0 [95% CI: 1.0, 1.1], p = 0.223) were associated with OS when adjusted by tumour grade and RSC but RSC was (95% CI: 0.2, 0.8, p = 0.012). CONCLUSION: Radiation dose to tumour during PRRT was predictive of radiologic response but not survival. Survival outcomes may relate to other biological factors. There was no evidence that MITVSSR change was associated with OS, but a larger study is needed.

[Diaphragmatic rupture and right ipsilateral intercostal hernia in chronic cough]. / Rupture diaphragmatique et hernie intercostale droites homolatérales sur toux aiguë.

INTRODUCTION: We are reporting the case of a 64-year-old patient with chronic cough who has been diagnosed with an intercostal hernia with pleural and hepatic content associated with a diaphragmatic hernia of non-traumatic origin. CASE REPORT: The patient was treated for an acutely febrile cough with signs of respiratory distress. Thoracic scan showed an intercostal hernia containing an encysted hematoma and a right anterior diaphragmatic hernia with epiploic content. The COVID PCR was negative. This is one of the rare reported cases of intercostal hernia associated with a homolateral diaphragmatic rupture. Visceral and thoracic surgery enabled treatment of the two hernial orifices by raphy as well as omentectomy of the necrotic omentum ascending to the right pulmonary hilum. CONCLUSION: These two parietal complications of chronic cough should be considered in case of intercostal flap or acute respiratory distress. Surgery must then be carried out as a matter of urgency to reduce the content of the hernias and treat the musculoaponeurotic dehiscent orifices.

Prediction and monitoring of relapse in stage III melanoma using circulating tumor DNA.

BACKGROUND: The advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma. PATIENTS AND METHODS: Somatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29). RESULTS: ctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5-5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3-24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3-5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3-27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status. CONCLUSION: Baseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.

A phantom for testing of 4D-CT for radiotherapy of small lesions.

PURPOSE: The use of time-resolved four-dimensional computed tomography (4D-CT) in radiotherapy requires strict quality assurance to ensure the accuracy of motion management protocols. The aim of this work was to design and test a phantom capable of large amplitude motion for use in 4D-CT, with particular interest in small lesions typical for stereotactic body radiotherapy. METHODS: The phantom of "see-saw" design is light weight, capable of including various sample materials and compatible with several surrogate marker signal acquisition systems. It is constructed of polymethylmethacrylate (Perspex) and its movement is controlled via a dc motor and drive wheel. It was tested using two CT scanners with different 4D acquisition methods: the Philips Brilliance Big Bore CT (helical scan, pressure belt) and a General Electric Discovery STE PET∕CT (axial scan, infrared marker). Amplitudes ranging from 1.5 to 6.0 cm and frequencies of up to 40 cycles per minute were used to study the effect of motion on image quality. Maximum intensity projections (MIPs), as well as average intensity projections (AIPs) of moving objects were investigated and their quality dependence on the number of phase reconstruction bins assessed. RESULTS: CT number discrepancies between moving and stationary objects were found to have no systematic dependence on amplitude, frequency, or specific interphase variability. MIP-delineated amplitudes of motion were found to match physical phantom amplitudes to within 2 mm for all motion scenarios tested. Objects undergoing large amplitude motions (>3.0 cm) were shown to cause artefacts in MIP and AIP projections when ten phase bins were assigned. This problem can be mitigated by increasing the number of phase bins in a 4D-CT scan. CONCLUSIONS: The phantom was found to be a suitable tool for evaluating the image quality of 4D-CT motion management technology, as well as providing a quality assurance tool for intercenter∕intervendor testing of commercial 4D-CT systems. When imaging objects with large amplitudes, the completeness criterion described here indicates the number of phase bins required to prevent missing data in MIPs and AIPs. This is most relevant for small lesions undergoing large motions.

A programmable motion phantom for quality assurance of motion management in radiotherapy.

A commercially available motion phantom (QUASAR, Modus Medical) was modified for programmable motion control with the aim of reproducing patient respiratory motion in one dimension in both the anterior-posterior and superior-inferior directions, as well as, providing controllable breath-hold and sinusoidal patterns for the testing of radiotherapy gating systems. In order to simulate realistic patient motion, the DC motor was replaced by a stepper motor. A separate 'chest-wall' motion platform was also designed to accommodate a variety of surrogate marker systems. The platform employs a second stepper motor that allows for the decoupling of the chest-wall and insert motion. The platform's accuracy was tested by replicating patient traces recorded with the Varian real-time position management (RPM) system and comparing the motion platform's recorded motion trace with the original patient data. Six lung cancer patient traces recorded with the RPM system were uploaded to the motion platform's in-house control software and subsequently replicated through the phantom motion platform. The phantom's motion profile was recorded with the RPM system and compared to the original patient data. Sinusoidal and breath-hold patterns were simulated with the motion platform and recorded with the RPM system to verify the systems potential for routine quality assurance of commercial radiotherapy gating systems. There was good correlation between replicated and actual patient data (P 0.003). Mean differences between the location of maxima in replicated and patient data-sets for six patients amounted to 0.034 cm with the corresponding minima mean equal to 0.010 cm. The upgraded motion phantom was found to replicate patient motion accurately as well as provide useful test patterns to aid in the quality assurance of motion management methods and technologies.

Motion effects on SUV and lesion volume in 3D and 4D PET scanning.

To assess the effect of lesion motion and respiration rate on Standardised Uptake Value (SUV) and the ability of 4D PET to restore any loss in SUV and distortion of lesion volume on two PET/CT systems. A Perspex phantom with four cylindrical reservoirs filled with (18)F-FDG was used in this study. The cylinders measured 5, 10, 15, and 20 mm in diameter. A GE Discovery STE8 (GE Medical Systems Milwaukee, WI) and a Siemens Biograph 64/40 (Siemens Medical Solutions, Erlangen, Germany) scanner was used to acquire a stationary un-gated PET scan of the phantom. Multiple 10 min list mode 4D PET scans were acquired using the Varian RPM on the GE camera and the Anzai Gating system on the Siemens camera. The phantom was scanned at five different respiratory rates and motion amplitudes in a sinusoidal fashion, 15 RPM/1 cm, 15 RPM/2 cm, 15 RPM/4 cm, 30 RPM/2 cm and 7.5 RPM/2 cm (RPM-respirations per minute). Each scan was reconstructed into ten bins and as an un-gated static image. The SUVmax, SUVmean and volume were measured for all four reservoirs using Siemens TrueD analysis software. With increasing lesion movement the SUVmax and SUVmean decreased and the volume increased with the SUVmax in the smallest lesion underestimated by up to a factor of four. The SUVmax, SUVmean and volume were mostly recovered using 4D imaging regardless of amount of lesion displacement. The larger lesions showed better count recovery and volume correction than the smaller lesions. The respiratory rate had no effect of SUV or volume. Un-gated imaging of moving lesions decreases apparent SUV in small lesions significantly and overestimates volumes. 4D PET scanning recovers most of the apparent loss in SUV and distortion of volumes.

A comparative study of cytoplasmic granules imaged by the real-time microscope, Nile Red and Filipin in fibroblasts from patients with lipid storage diseases.

Cytoplasmic granules in fibroblasts, visualized without stains, or labelled with Nile red, Filipin, or anti-LAMP-1 (lysosome-associated membrane protein 1), were imaged using the real-time microscope (RTM). New advances in light microscope technology were applied to detect cytoplasmic granules (RTM-visible granules) and characterize them by imaging contrast, size, shape, cellular distribution, composition, motion dynamics and quantity. Appearing as solid spheroids or ring structures, the majority of the RTM-visible granules contained Nile-red labelled neutral lipids. A smaller subpopulation, appearing dimmer, with less imaging contrast, contained Filipin-labelled free cholesterol. Most lipid storage granules have a diameter ranging from 0.3 mum to 0.6 mum, with a small population measuring up to 1 mum. They typically clustered in the perinuclear region and displayed relatively small oscillatory motion. Immunofluorescence based on LAMP-1 labelling highlighted granular structures that were distinct and separate from RTM-visible granules and other structures in the light modality of the microscope. RTM-visible granules were associated with disease phenotypes that have increased cellular neutral lipid stores corresponding to the Nile red-labelled droplets (e.g. triacylglycerides, cholesterol esters). As predicted, the fibroblast strains with a defect resulting in Wolman disease, when compared to control samples, consistently had RTM-visible granules, higher in imaging contrast and with larger diameters, that were labelled with Nile red, and also an increased frequency of Filipin-cholesterol complexes. By comparison, in fibroblasts where the lipid storage is less evident (Gaucher and Farber diseases) or from GM(1) gangliosidosis, where the primary storage substances are oligosaccharides, fewer and smaller RTM-visible granules were observed. In some cases, changes in contrast and morphology in the unstained cytoplasmic compartments were more evident than in the labelled structures. In summary, applying the RTM imaging system to fibroblasts enables differences between the various disease types to be seen and, in specific examples, a unique phenotype can be readily discerned.

Sulfatide and Na+-K+-ATPase: a salinity-sensitive relationship in the gill basolateral membrane of rainbow trout.

We investigated the effect of salinity on the relationship between Na(+)-K(+)-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na(+)-K(+)-ATPase activity, especially in Na(+)-K(+)-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na(+)-K(+)-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na(+)-K(+)-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na(+)-K(+)-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na(+)-K(+)-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.

Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542.

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).

Preparation of Fab' from murine IgG2a for thiol reactive conjugation.

Lysyl endopeptidase (LE) from Achromobacter lyticus M497-1 (EC 3.4.21.50) was utilized to prepare F(ab')2 fragments from mouse anti-P-glycoprotein IgG2a obtained from the UIC2 hybridoma. This report describes a novel single step purification procedure for F(ab')2 fragments that eliminates residual LE activity responsible for secondary cleavage of F(ab')2 to Fab fragments. The purification of F(ab')2 and Fc fragments was accomplished utilizing protein G affinity chromatography and either gradient or step changes in the pH/ionic strength for elution of the Fc and F(ab')2 fragments. Residual LE was eluted from the protein G column with buffer containing 200 mM L-lysine prior to elution of F(ab')2 and Fc fragments. The activity of LE was monitored using the fluorogenic substrate Boc-Val-Leu-Lys-7-amido 4-methyl coumarin. A similar purification procedure for F(ab')2 fragments produced following pepsin digestion of IgG2a is also outlined. The ability of Fab' fragments, from reduced F(ab')2 fragments following LE digestion of IgG2a, to conjugate to thiol reactive groups was demonstrated using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-meso chlorin e6 mono (N-2-aminoethylamide) (Mce6) conjugates containing reactive maleimide groups. The biological activity of the Fab' targeted HPMA copolymer-Mce6 conjugates was tested against the P-glycoprotein expressing human ovarian carcinoma A2780/AD cell line utilizing a cell survival assay. Fab' targeted HPMA copolymer-Mce6 conjugate demonstrated significantly higher cytotoxicity than either a monoclonal antibody (mAb) targeted HPMA copolymer-Mce6 conjugate or a non-targeted HPMA copolymer-Mce6 conjugate, p < 0.05.

Multiple gestations: some public policy issues.

Multiple gestations, or multifetal pregnancies, raise a number of significant policy questions concerning the well being of women and the well being of the children fetuses might become. Important questions for feminists pertain not only to multifetal pregnancy itself, but also to the medical interventions associated with these pregnancies. In this paper, we address the questions of how many embryos should be transferred in assisted reproduction, how many fetuses should remain in a multiple gestation, who should make these decisions, and the need to protect women from overexposure to exogenous hormones. Although we focus on assisted reproduction in the United States, we believe that our suggestions are applicable to other countries where the technology is comparable.

The impact of substance abuse treatment modality on birth weight and health care expenditures.

During the 1990s, substance abuse treatment programs were developed for pregnant women to help improve infant birth outcomes, reduce maternal drug dependency and promote positive lifestyle changes. This study compared the relative impact of five treatment modalities--residential, outpatient, residential/outpatient, methadone and detoxification-only--on infant birth weight and perinatal health care expenditures for a sample of 445 Medicaid-eligible pregnant women who received treatment in Massachusetts between 1992 and 1997. Costs and outcomes were measured using the Addiction Severity Index and data from birth certificates, substance abuse treatment records and Medicaid claims. Multiple regression was used to control for intake differences between the groups. Results showed a near linear relationship between birth weight and amount of treatment received. Women who received the most treatment (the residential/outpatient group) delivered infants who were 190 grams heavier than those who received the least treatment (the detoxification-only group) for an additional cost of $17,211. Outpatient programs were the most cost-effective option, increasing birth weight by 139 grams over detoxification-only for an investment of only $1,788 in additional health care and treatment costs. A second regression using five intermediate treatment outcomes--prenatal care, weight gain, relapse, tobacco use and infection--suggested that increases in birth weight were due primarily to improved nutrition and reduced drug use, behaviors which are perhaps more easily influenced in residential settings.

Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.

Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastin-binding protein.

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.

Novel peptidomimetic hematoregulatory compounds.

The activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore model inferred from the structure activity relationships of a peptide SK&F 107647 (1), is reported. These compounds induce a hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic agents in the area of hematoregulation.

Intracellular forms of human NOTCH1 functionally activate essential Epstein-Barr virus major latent promoters in the Burkitt's lymphoma BJAB cell line but repress these promoters in Jurkat cells.

We have demonstrated that intracellular forms of NOTCH1 transactivate two major Epstein-Barr virus (EBV) latent promoters, the LMP1 and Cp1 promoters in an EBV-negative B-cell line, BJAB. Truncated intracellular NOTCH1 associated with the nuclear membrane (DeltaE) transactivates the LMP1 promoter fivefold; however, the intranucleus localized form of NOTCH1 (ICN) transactivates this promoter approximately twofold in chloroamphenicol acetyltransferase (CAT) reporter assays in BJAB cells. Additionally, DeltaE activated the major Cp1 promoter 12-fold, whereas the ICN form of NOTCH1 activates at only about half that level when compared to that of DeltaE membrane-bound NOTCH1. This result differs from previously observed data, where intracellular NOTCH1 bound to the nuclear membrane, DeltaE, and nucleus-localized NOTCH1, ICN, all had similar levels of activation in 293 cells. This suggests distinct transcriptional activities in different cell types. Moreover, in Jurkat cells, a T-cell line, intranucleus localized NOTCH1 molecules demonstrated a repressive activity against the two EBV major latent promoters. Only DeltaE activated the Cp1 and LMP1 promoters at a level slightly above background, whereas intranucleus localized NOTCH1 ICN, or the form of NOTCH1 lacking the ankyrin repeats, DeltaE(TAR), surprisingly resulted in the repression of these promoters in Jurkat cells. Similarly, another truncated form of NOTCH1, referred to as ICNW, which contains the tryptophan residue W(1767) within one of the RBP-Jkappa interacting domains, repressed the LMP1 promoter approximately twofold. Further analysis of the truncated NOTCH1 molecules on the LMP1 promoter element, lacking the two RBP-Jkappa binding sites, suggests that repression in Jurkat cells may be affected by the presence of the two RBP-Jkappa binding sites. These studies indicate that intracellular NOTCH1 can activate the EBV major latent promoters in BJAB cells. However, in Jurkat cells, intracellular truncated forms of NOTCH1 lacking the RBP-Jkappa binding sites repress these EBV latent promoters. Only the membrane-bound form of NOTCH1, DeltaE, activated the EBV major latent promoters in Jurkat cells, albeit at a lower level than that seen in BJAB cells. Our data suggest that EBNA2 and truncated intracellular nuclear localized forms of NOTCH1 may be functionally similar in their interactions with RBP-Jkappa; however, these molecules may have distinctly different transcriptional partners in BJAB and Jurkat cells. Moreover, these truncated NOTCH1 molecules may not represent the normal processed forms of NOTCH1 in cells and may exhibit dominant negative phenotypes in the absence of the required posttranslational modifications. Further investigations are necessary to determine the similarity and differences occurring with intracellular NOTCH1 in other B- and T-cell lines.

Intracellular forms of human NOTCH1 interact at distinctly different levels with RBP-jkappa in human B and T cells.

The cellular transcriptional repressor RBP-Jkappa associates with the Epstein-Barr virus nuclear antigens (EBNAs) determined to be essential for transformation of human primary B lymphocytes. It was demonstrated through genetic analysis that interaction between the viral transactivator EBNA2 and RBP-Jkappa is essential for EBV immortalization of primary B lymphocytes. We have shown that the association of RBP-Jkappa with intracellular NOTCH1 differs significantly in B and T cells. Immunoprecipitation analyses with antibodies to both the intracellular forms of NOTCH1 and to RBP-Jkappa demonstrated that little or no RBP-Jkappa is associated with NOTCH1 in B cell lines compared to the RBP-Jkappa associated with NOTCH1 in T cell lines and was further demonstrated in human primary lymphocytes. Additionally, EBNA2 can compete with intracellular NOTCH1 for binding to GST-RBP-Jkappa in vitro. Northern blot for the cellular gene hairy enhancer of split (HES1) demonstrated that HES1 is upregulated in the EBV transformed lymphoblastoid cells expressing high levels of EBNA2 and in a T cell line SupT1 overexpressing intracellular activated NOTCH1. Hence, EBNA2 may be able to compete for the available pool of RBP-Jkappa more effectively in human B cells than in T cells and provides a possible explanation for the ability of EBV to potently and efficiently infect and immortalize human B cells. Leukemia (2000) 14, 84-92.

Hepatitis C in Peru: risk factors for infection, potential iatrogenic transmission, and genotype distribution.

A large seroepidemiologic and genotyping study of hepatitis C virus (HCV) was conducted in Lima, Peru, during the periods of 1986 to 1993 (cohort A) and 1994 (cohort B). Anti-HCV seroprevalence rates were 15.6% (216 of 1,389) and 11.7% (168 of 1,438), respectively. Low rates were seen among volunteer blood donors (1.1% and 0.8%). Anti-HCV rates were much higher among patients undergoing hemodialysis (43.7% and 59.3%), hemophiliacs (60.0% and 83.3%), in those more than 39 years old (18.2% and 26.0%), in females (25.0% and 27.4%), and in less-educated persons (16.9%). Age- and gender-adjusted risk factors in cohort B included blood transfusion history (adjusted odds ratio [AOR] = 29.8), prior organ transplantation (AOR = 9.1) or a history of hepatitis (AOR = 4.9), previous hospitalization (AOR = 3.7), a history of intravenous drug use (AOR = 3.5), prior major surgery (AOR = 2.6), a history of acupuncture (AOR = 2.1), previous dental procedures (AOR = 1.2), and prior medical injections (AOR = 1.04). The most prevalent HCV genotype was type 1 (86%), followed by type 3 (10%) and type 2 (2%). Transmission through unsafe injection-related and medical/dental procedures appears to play an important role in HCV infection among Peruvians.

Distinct patterns of viral antigen expression in Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus coinfected body-cavity-based lymphoma cell lines: potential switches in latent gene expression due to coinfection.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens approximately 110 and 41-42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts.